Purification and functional characterization of a low-molecular-mass Ca2+,Mg2+- and Ca2+-ATPase modulator protein from rat brain cytosol.
A low-molecular-mass modulator protein having a molecular mass of about 12 kDa has been purified from rat brain cytosol following gel filtration and FPLC/Mono Q anion-exchange chromatographic separation. A number of protein fractions were obtained from an FPLC column when eluted with a 0.1 M NaCl hold gradient. One fraction (peak no. 5) was found to stimulate Ca2+,Mg2+-ATPase but inhibit Ca2+-ATPase isolated from goat spermatozoa. The S50 (concentration producing 50% stimulation) and I50 were found to be in the nanomolar range. The modulator seems to bind to Ca2+, Mg2+- or Ca2+-ATPase at a site distal from the ATP binding site. The binding to both the ATPases is reversible and non-competitive in nature. The inhibitory activity is found to depend significantly on -SH or -NH2 group(s) of the modulator, whereas no appreciable dependency of the stimulatory effect was apparent. The study indicates that the modulator is not a glycoprotein. CD analysis suggests that the protein exists as an unordered secondary structure. An immuno-cross-reactivity study with specific antibody and inhibition by thapsigargin suggests that the Ca2+,Mg2+- and Ca2+-ATPases from goat testes microsomal membranes are two isoforms of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA) family. The modulator does not contain any Trp molecules, as evident from Trp fluorescence analysis. Amino acid analysis shows that glycine, serine, derivatives of tyrosine and phenylalanine are the predominant amino acids. The data suggest that the modulator is a negatively charged protein and is a good tool for distinguishing the regulation of Ca2+,Mg2+- and Ca2+-ATPase activities.
Bhattacharyya
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Department of Chemistry, Bose Institute, 93/1, A.P.C. Road, Calcutta 700 009, India.
Sen
P C
PC
D000818:Animals; D001831:Body Temperature; D001921:Brain / Q000201:enzymology*; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002942:Circular Dichroism; D003600:Cytosol / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006041:Goats; D008297:Male; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013094:Spermatozoa / Q000201:enzymology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry; D014700:Verapamil / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D013439:Sulfhydryl Reagents; D014268:Trifluoperazine; D014364:Tryptophan; D014700:Verapamil; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases